The Studies On Preparation And Properties Of Antimicrobial Peptides From Wheat Gluten

Wheat is one of the major food crops in China，its annual processed capacity is up to100million tons．As a by-product of starch industry，wheat protein distributes widely and is richin resource．In-depth study of the wheat protein can enhance its economic value and socialbenefits．In this paper，wheat protein (wheat gluten) as raw materials，specific protease enzymewere selected to prepare antibacterial peptide．Study involved optimization of processconditions on the enzymatic hydrolysis，hydrolysates refined，the functional properties andstructure of antibacterial peptide．Initially，the preparation conditions of antimicrobial peptide were researched．Alcalaseand Protamex protease were selected to prepare two different peptides through comparing theinhibitory spectrum and activity．The optimum enzymolysis conditions were obtained byorthogonal tests based on Single factor analysis．The optimal enzymolysis conditions ofAlcalase was that the hydrolysis time90min，the amount of enzyme2100U/g，pH8.5，temperature55℃，the substrate concentration3%，Under the conditions，the averageinhibition rate of the two bacteria (Escherichia coli and Staphylococcus aureus) was8.9%．The best process of Protamex protease as follows：temperature55℃， pH9.0，enzymedosage of1800U/g，substrate concentration2.0%，Under these conditions，the averageinhibition rate of the two bacteria (Escherichia coli and Staphylococcus aureus) and two fungi(Aspergillus niger and Aspergillus) was9.6%．The separation of peptide A and P was followed by ultrafiltration，Sephadex G-25chromatography and RP-HPLC method，respectively．The results of ultrafiltration showedthat the peptide A and P fraction of the relative molecular mass less than5KDa(Called A-cand P-c component，respectively) had inhibition activity significantly，And then by SephadexG-25gel chromatography，A-c peptide was separed to four peaks，in which the peakⅡ(relative molecular weight2036Da) had the best inhibition activity，the inhibition rate of E.coli was20.6%，the inhibition rate of Staphylococcus aureus was19.1%，P-c peptide fractionswas separed to five peaks，the peak Ⅲ(relative molecular mass1721Da) was of the strongestinhibitory effect，the inhibition rate of E. coli and Staphylococcus aureus were19.6%，18.2%，respectively， the inhibition rate of Aspergillus niger were22.1%，the aflatoxin was18.9%．Four components have been obtained from A-c-Ⅱby RP-HPLC separation，whichpeaks3(referred to as A-c-Ⅱ-3) had the strongest inhibitory effect，the inhibition rate of E. coli reached53.8%，Staphylococcus aureus51.6%．Three components have been obtainedfrom P-c-Ⅲ by RP-HPLC separation，which peak3(referred to as P-c-Ⅲ-3) had thestrongest inhibitory effect，the inhibition rate of E. coli and Staphylococcus aureus reached51.8%，49.1%，respectively，and the inhibition rate of Aspergillus niger，Aspergillus flavusreached54.3%，51.2%，respectively．The structure of antimicrobial peptides have been identified by amino acid compositionand mass spectrometry．MS/MS and amino acid analysis showed that the relative molecularmass of antimicrobial peptide Ac-Ⅱ-3was2080.2Da，the amino acid sequence of A-c-Ⅱ-3was Arg－Thr－Thr－Thr－Leu－Arg－Lys－Ser－Pro－Cys－Pro－Leu－Val－Cys－Phe－Gly－Gly－Met－Asp．the relative molecular weight of P-c-Ⅲ-3was1764.9Da andthe amino acid sequence of P-c-Ⅲ-3was Tyr－Leu－His－Phe－Lys－Arg－Asp－Leu－Cys－Cys－Val－Gly－Gly－Thr－Arg．Antimicrobial peptides was studied on the stability of temperature，pH，ion concentrationand minimal inhibitory concentration(MIC)．The results showed that pH influence on theactivity of antimicrobial peptide，antimicrobial peptides had the higher antibacterial activity inpH8；under the conditions of freezing and boiling，antimicrobial peptides would decreaseactivity；the presence of ions would affect the activity of antimicrobial peptides，Theantibacterial activity had a decreasing trend with the ion concentration increasing；Theminimal inhibitory concentration (MIC) of Antimicrobial peptide A-c-Ⅱ-3and P-c-Ⅲ-3against Escherichia coli， Staphylococcus aureus， Salmonella， Aspergillus niger andAspergillus was in32-59μg/ml．...